Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota

The Effective Fragment Molecular Orbital Method for Fragments Connected by Covalent Bonds

We extend the effective fragment molecular orbital method (EFMO) into treating fragments connected by covalent bonds. The accuracy of EFMO is compared to FMO and conventional ab initio electronic structure methods for polypeptides including proteins. Errors in energy for RHF and MP2 are within 2 kcal/mol for neutral polypeptides and 6 kcal/mol for charged polypeptides similar to FMO but obtained two to five times faster. For proteins, the errors are also within a few kcal/mol of the FMO results. We developed both the RHF and MP2 gradient for EFMO. Compared to ab initio, the EFMO optimized structures had an RMSD of 0.40 and 0.44 {\AA} for RHF and MP2, respectively.Comment: Revised manuscrip

Inactivation of murine norovirus by chemical biocides on stainless steel

<p>Abstract</p> <p>Background</p> <p>Human norovirus (NoV) causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV) as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces.</p> <p>Methods</p> <p>The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA) or glutaraldehyde (GDA) for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells.</p> <p>Results</p> <p>PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v)] and 1-propanol [30% (v/v)] were able to inactivate MNV under clean conditions (0.03% BSA) on the carriers by ≥ 4 log₁₀within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v). Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes).</p> <p>Conclusion</p> <p>Products based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.</p

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

BioOK – a Comprehensive System for Analysis and Risk Assessment of Genetically Modified Plants

Gentechnisch veränderte (GV) Pflanzen müssen im Rahmen des Zulassungsverfahrens in der EU auf ihre potentiellen Auswirkungen auf die Umwelt und die mensch­liche oder tierische Gesundheit analysiert werden. Der gegenwärtige Zulassungsprozess ist ein Konglo­merat verschiedenster Analysemethoden und extrem zeit- und kostenaufwendig. Das Anliegen von BioOK als ein multidisziplinäres wissenschaftliches Netzwerk ist die Entwicklung von maßgeschneiderten Ansätzen zur Risikoanalyse von GV Pflanzen auf der Grundlage von Ursache-Wirkungs­hypothesen mit dem Ziel des Aufbaus eines effektiven und qualifizierten Risikobewertungssystems. Die Forschungsaktivitäten von BioOK zielen auf einen Paradigmenwechsel im aktuellen Zulassungsprozess. Sie basieren auf einem modularen System, das alle Aspekte des Risikomanagements umfasst: molekulare Charakterisierung, Inhaltsstoffanalyse, agronomische Eigenschaften, Ziel- und Nichtzielorganismen, Boden und Mikroorganismen, Toxikologie, Allergenität und Überwachung nach Markt­einführung, wobei jeder Modul unterschiedliche Analysemethoden beinhaltet. Die durch BioOK angestrebte Reform des Risikobewertungsprozesses von GV Pflanzen umfasst zwei Phasen: zunächst die Optimierung der Analysemethoden selbst und dann die Etablierung eines Entscheidungsunterstützungssystems (Test Decision System – DSS), basierend auf biologischen Schwankungsbreiten (baselines), Zeigermerkmalen (indicators) und Grenzwerten (thresholds) für jede Analysemethode. BioOK hat in einer ersten Entwicklungsphase bereits optimierte Testmethoden entwickelt: Für die Inhaltsstoffanalyse wurde die Untersuchung auf substantielle Äquivalenz durch GC-MS, LC-MS und HPLC/RI Methoden vereinfacht. Ein neu eingeführtes Analyseschema zur Ermittlung potentieller Effekte von GV Pflanzen auf den Boden kombiniert ein in vitro System zur Beprobung von Rhizodepositaten von Pflanzen, die unter kontrollierten Umweltbedingen gewachsen sind, sowie die entsprechenden Bodentypen und deren Charakterisierung mit offenen und hochsensitiven molekular-chemischen Screening und Fingerprinting-Methoden. Ein neues in vitro System zur Simulation des Transports von Substanzen aus dem Darm ins Blut, das das Risiko der Aufnahme durch Mensch oder Tier zu einem frühen Zeitpunkt misst, wurde entwickelt. Um die Effektivität und Reproduzierbarkeit von Probenahmen an der Pflanze zu erhöhen, wird ein genau definiertes Probenahmeschema entwickelt. Schließlich, in Ergänzung der aktuellen Methodik zur Allgemeinen Überwachung (General Surveillance) von GV Pflanzen im Anbau, wurde eine Herangehensweise zur Abschätzung der Notwendigkeit für ein europaweites fallspezifisches (Case Specific) Monitoring beruhend auf Ursache-Wirkungsszenarien, erarbeitet. Die zweite Phase der BioOK F&amp;E-Arbeiten konzentriert sich auf die Entwicklung eines Entscheidungsunterstützungssystems (Decision Support System, DSS). Dazu wird ein computergestütztes System implementiert, in dem alle standardisierten und validierten Methoden zu einem Entscheidungsbaum mit Knotenpunkten, definiert über biologische Schwankungsbreiten und potentielle Risiken definierenden Grenzwerten für Zeigermerkmale, zusammengeführt sind. &nbsp; &nbsp;Genetically modified (GM) plants have to be analyzed for their potential impacts on the environment and on human or animal health before authorisation by the EU. The approval process currently refers to a conglomeration of diverse analytical methods and is intensive in time and costs. The intention of BioOK as a multidisciplinary scientific network is the development of tailor-made approaches for GM plants based on a cause-effect hypothesis to obtain an effective and qualified risk assessment system. The research activity of BioOK aims to renew the current approval process. It is based on a modular system covering all aspects of risk assessment: molecular characterisation, compound analysis, agronomic traits, target and non-target organisms, soil and micro organisms, toxicology, allergenicity and post-market monitoring, each module containing several test methods. The renewal of the risk assessment procedure intended by BioOK consists of two phases: first the optimization of test methods and second the establishment of a decision support system (DSS) based on baselines, indicators and thresholds developed for each of the methods. Optimized test methods have been developed mainly during the first phase: For compound analysis methods have been developed to ease the analysis of substantial equivalence of the events by GC-MS, LC-MS and HPLC/RI. A newly introduced testing scheme for the detection of potential effects of GM plants on soil combines an in-vitro system to collect rhizodeposits from plants grown under controlled environmental conditions and the correspon­ding bulk soil, and their characterisation by untargeted and highly sensitive molecular-chemical screening and fingerprinting technique. A novel in vitro system simula­ting the transport of substances from the gut into the blood that detects the risk of incorporation in human or animal at an early time point was developed. In order to increase the effectiveness and reproducibility of the sampling procedure we developed a valid defined sampling scheme. Finally, complementing the actual General Surveillance methodology, an approach for a Europe-wide case specific monitoring referring to cause-effect sce­narios was developed. The second phase concentrates on the development of a Decision Support System (DSS). A computer-based system will implement and merge all standardized methods in a decision tree system following decision rules defined by baseline and thresholds for indicators. &nbsp; &nbsp

German Multicenter Study Analyzing Antimicrobial Activity of Ceftazidime-Avibactam of Clinical Meropenem-Resistant Pseudomonas aeruginosa Isolates Using a Commercially Available Broth Microdilution Assay

Multidrug resistance is an emerging healthcare issue, especially concerning Pseudomonas aeruginosa. In this multicenter study, P. aeruginosa isolates with resistance against meropenem detected by routine methods were collected and tested for carbapenemase production and susceptibility against ceftazidime-avibactam. Meropenem-resistant isolates of P. aeruginosa from various clinical materials were collected at 11 tertiary care hospitals in Germany from 2017–2019. Minimum inhibitory concentrations (MICs) were determined via microdilution plates (MICRONAUT-S) of ceftazidime-avibactam and meropenem at each center. Detection of the presence of carbapenemases was performed by PCR or immunochromatography. For meropenem-resistant isolates (n = 448), the MIC range of ceftazidime-avibactam was 0.25–128 mg/L, MIC90 was 128 mg/L and MIC50 was 16 mg/L. According to EUCAST clinical breakpoints, 213 of all meropenem-resistant P. aeruginosa isolates were categorized as susceptible (47.5%) to ceftazidime-avibactam. Metallo-β-lactamases (MBL) could be detected in 122 isolates (27.3%). The MIC range of ceftazidime-avibactam in MBL-positive isolates was 4–128 mg/L, MIC90 was >128 mg/L and MIC50 was 32 mg/L. There was strong variation in the prevalence of MBL-positive isolates among centers. Our in vitro results support ceftazidimeavibactam as a treatment option against infections caused by meropenem-resistant, MBL-negative P. aeruginosa

Phenotypic and transcriptomic analyses of seven clinical Stenotrophomonas maltophilia isolates identify a small set of shared and commonly regulated genes involved in the biofilm lifestyle

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings. IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms

MixInYeast: A Multicenter Study on Mixed Yeast Infections

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions